lecture 23: metabolomics technologywsu.edu/~lange-m/documnets/lange_metabolomicstechnologies.pdf ·...
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MBioS 478/578 Bioinformatics Mark Lange
Lecture 23: Metabolomics Technology
•Definitions and Background
•Technologies•Nuclear Magnetic Resonance•Mass Spectrometry – General Introduction•Fourier-Transform Mass Spectrometry•Gas Chromatography – Mass Spectrometry•Liquid Chromatography – Mass Spectrometry•Capillary Electrophoresis – Mass Spectrometry
•Applications
MBioS 478/578 Bioinformatics Mark Lange
Definitions and Background
The metabolome represents the collection of all metabolites in a biological organism, which are the end products of its gene expression. http://en.wikipedia.org/wiki/Metabolome
First use of the term "metabolome" in the literature:Oliver, S.G., Winson, M.K., Kell, D.B., Baganz, F. (1998). Systematic functional analysis of the yeast genome, Trends Biotechnol. 16(10): 373–378.
Metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" - specifically, the study of their small-molecule metabolite profiles http://en.wikipedia.org/wiki/Metabolomics
The word metabonomics is also used, particularly in the context of drug toxicity assessment or when NMR-based techniques are used. There is some disagreement over the exact differences between 'metabolomics' and 'metabonomics'; in general, the term 'metabolomics' is more commonly used.http://en.wikipedia.org/wiki/Metabolomics
MBioS 478/578 Bioinformatics Mark Lange
Definitions and Background
For investigators of selected biochemical pathways, it is also often not necessary to view the effects of perturbation on all branches of metabolism. Instead, the analytical procedure can be focused on a smaller number of pre- defined metabolites. Sample preparation and data acquisition can be focused on the chemical properties of these compounds with the chance to reduce matrix effects. This process is called “metabolite profiling” (or sometimes “metabolic profiling”). http://www.genomicglossaries.com/content/printpage.asp?REF=/content/metabolic_engineering.asp
For functional genomic or plant breeding programmes, as well as for diagnostic usage in industrial or clinical routines, it might not be necessary to determine the levels of all metabolites individually. Instead, a rapid classification of samples according to their origin or their biological relevance might be more adequate in order to maintain a high throughput. This process can be called “metabolic fingerprinting”.http://www.genomicglossaries.com/content/printpage.asp?REF=/content/metabolic_engineering.asp
General problems encountered when characterizing the metabolome are the highly complex nature and the enormous chemical diversity of physiologically relevant metabolites.
At present, the metabolome cannot be computed from the genome. (Trethewey, R.N. (2004) Curr. Opin. Plant Biol. 7, 196-201)
It has been estimated that plants produce more than 200,000 metabolites.(Fiehn, O. (2002) Plant Mol. Biol. 48, 155-171)
Role of post-genomic technologies in systems biology(Nicholson, Connelly, Lindon, Holmes Nature Rev. Drug Discov. 1, 153 – 161)
Real World
Inputs:Noxious agent
StressorMutation
Outputs:Biological end-points
Pathology
‘Omics World’
Gene expression
Protein profile
Metabolic profile
Real world end-point
Indicator for end-point
Indicator/ end-point
Integrative analysis of genes, proteins and metabolites(Fiehn O, Weckwerth W. 2003. Deciphering metabolic networks. Eur. J. Biochem. 270, 579–588)
Problems:
No single extraction procedure works for all metabolites because conditions that stabilize one type of compound will destroy other types or interfere with their analysis.
Examples: nucleotides (ATP, S-adenosylmethionine, etc) are extracted using perchloric acid, a strong acid and oxidizing agent that attacks other compounds; alkaloids are extracted in alkaline media that destroy aldehydic compounds; thiols and 2-oxo acids are so labile that they must be extracted using aggressive reagents that convert them to stable derivatives but which interfere with determination of other analytes.
Therefore the extraction protocol has to be tailored to the metabolites to be profiled.
In practice, these considerations mean that metabolic profiling has up to now largely been confined to fairly stable compounds that can be extracted together. These include major primary metabolites (sugars, sugar phosphates, amino acids, and organic acids) and certain secondary metabolites (e.g., phenylpropanoids, alkaloids).
The most comprehensive profiling covers a few hundred such compounds, the majority of which remain unidentified. Many crucial metabolites are therefore currently being missed.
http://www.hos.ufl.edu/meteng/HOS6231-2002/METABOLICPROFILINGANDMETABOLOMICS2005.htm
MBioS 478/578 Bioinformatics Mark Lange
Technologies
Nuclear Magnetic Resonance
Advantages
•Can be coupled with liquid chromatography (increased resolution)
•Applicable with a wide range of metabolites
•Can be used to determine structure of metabolites
Disadvantages
•Time-consuming for most nuclei
•Relatively insensitive – not suited for low abundance compounds
MBioS 478/578 Bioinformatics Mark Lange
Technologies
Mass Spectrometry – General Introduction
(http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm)
MBioS 478/578 Bioinformatics Mark Lange
Technologies
Fourier-Transform Mass Spectrometry
Advantages
•Crude extracts can be injected directly (no chromatography separation)
•Very fast fingerprinting method
•Capable of ultra-high resolution (permits mass to be determined to four or five decimal places
•Empirical molecular formulae can be assigned to peaks
Disadvantages
•$ $ $
•High maintenance
•Inability to distinguish isomers of the same molecular mass
Fourier-Transform Mass Spectrometry(http://www.bumc.bu.edu/Dept/Content.aspx?DepartmentID=385&PageID=7205)
MBioS 478/578 Bioinformatics Mark Lange
Technologies
Gas Chromatography – Mass Spectrometry
Advantages
•Very high chromatographic resolving power
•Good selection of stationary phases
•Readily interfaced to a mass spectrometer
Disadvantages
•Compounds must be sufficiently volatile
•Compounds must be thermally stable
•Limited to nonpolar and slightly polar molecules
MBioS 478/578 Bioinformatics Mark Lange
Technologies
Liquid Chromatography – Mass Spectrometry
Advantages
•Capable of analyzing a wide range of metabolites (thermally labile, high polarity, high molecular mass)
•Good selection of stationary phases
•Readily interfaced to a mass spectrometer
Disadvantages
•Fragmentation rules not well established
•MS libraries missing
•Limited resolution (new developments: UPLC, chip-LC)
MBioS 478/578 Bioinformatics Mark Lange
Technologies
Capillary Electrophoresis – Mass Spectrometry
Advantages
•Short analysis times
•High separation efficiency
•Enables chiral separations
Disadvantages
•Methods development difficult
•MS libraries missing
Nature Biotechnology 18, 1157 - 1161 (2000)
Metabolite profiling for plant functional genomics
Oliver Fiehn, Joachim Kopka, Peter Dörmann, Thomas Altmann, Richard N. Trethewey & Lothar Willmitzer
Nature Biotechnology 18, 1157 - 1161 (2000)
Metabolite profiling for plant functional genomics
Oliver Fiehn, Joachim Kopka, Peter Dörmann, Thomas Altmann, Richard N. Trethewey & Lothar Willmitzer
Comprehensive chemical derivatization for gas chromatography–mass spectrometry-based multi-targeted profiling of the major phytohormones
J. Chromatography A 993, 89-102 (2003)
Claudia Birkemeyer, Ania Kolasa and Joachim Kopka
ABA
Cytokinin
IAA
SA
MJA
GA
Anal. Chem., 77 (10), 3090 -3100, 2005
Automated 20 kpsi RPLC-MS and MS/MS with Chromatographic Peak Capacities of 1000-1500 and Capabilities in Proteomics and Metabolomics
Y. Shen, R. Zhang, R.J. Moore, J. Kim, T.O. Metz, K.K. Hixson, R. Zhao, E.A. Livesay, H.R. Udseth, R.D. Smith